The present invention relates to an in-vitro method for prognosticating the illness development of patients with carcinoma of the breast and/or for diagnosing carcinoma of the breast, to kits suitable for performing said method, and to the use of T1-specific antibodies or fragments thereof or of T1-specific oligonucleotides for determining T1 protein or T1-mRNA in patients with carcinoma of the breast.
Carcinomas of the breast, in particular invasive carcinomas of the breast, are malign tumors with an extremely different clinical development that could so far not be predicted. Invasive carcinoma of the breast is the most frequently observed malign tumor in women; on the statistical average, every 16th woman suffers therefrom. In the case of an existing carcinoma of the breast, the breast tumor is first of all removed as the primary therapeutic measure. Especially the nodal state, but also tumor size, histological type, degree of differentiation and hormone receptor condition are nowadays regarded as important parameters for prognosis and further therapeutic planning. Emphasis is however placed on the evaluation of the state of the axillary lymph node. For instance with patients suffering from lymph node disease in the axillary region (nodal-positive, N(+)) already at the time of tumor diagnosis or surgery, a chemotherapy is normally carried out immediately, optionally supported by an additional radiation therapy. In general, patients with a negative axillary lymph node state (nodal-negative, N(0)) have a better chance and, therefore, are in general not subjected to a chemotherapeutic or radiation-therapeutic aftertreatment. Statistically, however, up to 30% of the patients classified as N(0) suffer from a relapse (Yan 1992). Such a high rate of relapse demonstrates that the factors of prognosis that have so far been known describe the illness development in a very incomplete manner only.
In recent years a great number of molecules were tested as to their possible use as prognosis factors for carcinoma of the breast (for the purpose of survey: Schmitt et al., 1994; Hoskins and Weber 1995), e.g. (proto)oncogenes such as c-erbB-2 (Allred et al., 1992, Archer et al., 1995), the tumor suppressor gene p53 (Barnes et al., 1993, Lipponen et al., 1993), the urokinase-type plasminogen activator uPA (Jaenicke et al., 1993; Wilhelm et al., 1994), the adhesion molecule E-cadherin (Rasbridge et al., 1993; Graff et al., 1995) and the cytoskeleton protein vimentin (Sommers et al., 1992). The vimentin synthesis correlates with the invasive growth of breast-carcinoma cell lines in vitro (Thompson et al., 1991) and partly with rapidly growing invasive ductal breast carcinomas having a poor prognosis in vivo (Domagala et al., 1990). Furthermore, possible factors of prognosis are formed by cytometrically determined morphometric features and texture features and also by DNA parameters (Auer et al., 1994).
However, none of the above-mentioned factors permits a sufficiently reliable prognosis of the further illness development after removal of the breast carcinoma.
It is therefore the object of the present invention to provide a method which allows for a sufficiently reliable prognosis of the further illness development in patients with carcinoma of the breast.
According to the invention this object is achieved by an in-vitro method for prognosticating the illness development of patients with carcinoma of the breast and/or for diagnosing carcinoma of the breast, which method comprises the qualitative or quantitative determination of T1 protein and/or T1-mRNA in sample material obtained from patients.
The T1 protein is an extracellular, soluble glycoprotein of 60-70 kDa (Werenskiold, 1992) with homology to members of the immunoglobulin superfamily, in particular the carcinoma-embryonal antigen (Klemenz et al., 1989).
The molecule was identified in an analysis of the former effects of oncoproteins (p21ras and p39v-mos) on the gene expression in fibroblasts (Werenskiold et al., 1989). In the mouse there is a synthesis of the T1 protein in embryonal tissues only; it cannot be detected in the adult animal (Rxc3x6xcex2ler et al., 1995 a,b). The function of the T1 protein has not been completely darified yet, but the isolation of a second membrane-bound variant of the molecule (T1-M) points to a function as a cytokine receptor. The membrane-bound T1-M protein is very similar to the IL-1 receptor type 1, but has no affinity to the cytokines IL-1 xcex1 and xcex2 (Rxc3x6xcex2ler et al, 1995 b; Danescu and Werenskiold, 1995) or IL-1 ra (Gayle et al., 1996). T1-M is a novel mast cell-specific cytokine receptor (Rxc3x6xcex2ler et al., 1995 b; Thomassen et al., 1995). The oncogene-inducible soluble variant of the T1 protein is a shortened form of said receptor and corresponds to the ligand-binding domain thereof. Recombinantly produced, soluble T1 from the mouse (Rupp et al., 1995) blocks the growth of mast cells.
So far, in breast carcimonas of the mouse, an overexpression of the soluble T1 protein has exclusively been observed in invasively growing, poorly differentiated tumors. Both the tumor stroma and the anaplastic tumor cells synthetize T1. In-situ hybridizations demonstrate an increased expression of T1 in tumor cells on the periphery of tumor cell complexes and, possibly induced by the tumor cells, in the stroma cells directly adjacent thereto (Rxc3x6xcex2ler et al, 1993). The induction of the T1 expression in the tumor cells correlates with the phenotypic transformation thereof and is accompanied by a loss in the E-cadherin and cytokeratin production. It is controlled by an AP1-dependent promoter of the T1 gene which is only active in non-hemopoietic (e.g. fibroblastic) cells (Thomassen et al., 1995). During phenotypic transformation of the epithelial tumor cells the induction of T1 is distinctly performed prior to that of the also AP1-dependent, mesenchymal cytoskeleton protein vimentin and therefore forms an early marker for the transformation process.
Surprisingly enough, it has now been found that the presence or absence of a T1 transcription or expression provides information about the future illness development, in particular information about the probability of the occurrence of a relapse or of the development or growth of metastases. As will be explained in detail in the examples, a high T1 value surprisingly correlates with a positive prognosis in patients with an N(0) nodal state whereas a high T1 protein or T1-mRNA level means a negative prognosis in patients with an N(+) nodal state.
In a preferred embodiment the T1 protein and/or the T1-mRNA is determined in a tumor tissue sample of the patient. To this end tissue sections are e.g. made and fixed in accordance with standard methods and are subsequently subjected either to an immunoassay for detecting an existing T1 protein or to hybridization with oligonucleotides, which are or can be labeled, or with DNA fragments. Total RNA or poly(A)+-mRNA can optionally be isolated from the tumor tissue according to standard methods and, for example after a gel-electrophoretic separation or after fixation to a solid matrix, the total RNA or poly(A)+-mRNA can then be determined again by hybridization with an oligonucleotide which is or can be labeled.
T1 protein can be detected not only in the tumor tissue itself, but also in various body fluids of the patients afflicted. In a preferred embodiment the method of the invention is carried out with a blood or serum sample:
There are a number of methods for determining T1-mRNA. As has already been pointed out above, it is possible, on the one hand, to detect T1-mRNA in situ or in corresponding RNA or mRNA preparations by hybridization with a corresponding oligonucleotide probe or a DNA fragment. The oligonucleotide probe or the DNA fragment itself can produce a measurable signal, i.e. it may be radioactively labeled, or can be capable of producing a signal by interacting with other molecules. For an improved evaluation of the corresponding tests the signal should be amplified in most cases. The target nucleic acid to be detected is normally amplified for this purpose.
For the amplification of the target sequence, i.e. T1-mRNA, by the PCR method (polymerase chain reaction) a cDNA copy is first of all made according to known methods. Said cDNA copy is then subjected to a PCR method. The sequence of the human T1 gene is already known in part (Tominaga et al., 1992); the selection of suitable oligonucleotides for performing the reverse transcription and the PCR method is thus within the scope of expert skill. An example of a suitable oligonucleotide is the oligonucleotide with the sequence 5xe2x80x2-CTT TGA TCA CCT GAA CTT TCT CTA GCA-3xe2x80x2 (SEQ ID NO:1) or a fragment thereof. A further suitable primer is the antisense primer 5xe2x80x2-AGT TTT CGG TTG TTG GTG CAT TTC-3xe2x80x2 (SEQ ID NO:2) or a suitable fragment thereof. Preferred primers derive from the 3xe2x80x2-untranslated region and/or the region of the exons 8 and 9. Primers from said regions have the advantage that they only hybridize with the RNA coding for the tumor-associated T1-S protein, but not with the T1-M-mRNA obtained by alternative splicing. One of the specific primers, sense or antisense, can optionally be replaced by a commercially available random primer. A number of further processes by which the target nucleic acids can be amplified have become known in the prior art in the meantime. Reference is here e.g. made to the Q-NASBA method in which the mRNA existing in the sample is amplified by the concerted action of reverse transcriptase, RNase H and T7 polymerase (Kievits et al., 1991). A further possibility is the detection of DNA obtained after reverse transcription through the so-called xe2x80x9cstrand displacement amplificationxe2x80x9d (SDA) (Walker et al., 1996, and the literature cited therein). One skilled in the art is also aware of a number of further methods which can also be used for detecting or determining T1-mRNA.
The amplification products formed are detected in a manner known to one skilled in the art. For instance, the DNA synthetized with the PCR method can be made visible by the incorporation of digoxigenin-containing nucleotides and subsequent reaction with enzyme-conjugated anti-digoxigenin antibodies. Any enzyme capable of producing a signal can be conjugated with the anti-digoxigenin antibody, for instance alkaline phosphatase, acid phosphatase, peroxidase, xcex2-D-galactosidase, glucose oxidase and horse-radish peroxidase. In response to the substrate used, the T1-mRNA can be detected quantitatively, e.g. by measuring the absorption or fluoresence of soluble products, or at least qualitatively.
For instance, the anti-digoxigenin antibody conjugated with alkaline phosphatase, which is obtainable from Boehringer Mannheim, is well suited for determining the existing T1-mRNA. One skilled in the art is aware that there are still further possibilities of detecting amplified nucleic acids, e.g. by incorporation of biotin-labeled nucleotides and subsequent reaction of the products with avidin- or streptavidin-conjugated enzymes which make it possible to produce a signal. Finally, such an enzyme may also be coupled to a third oligonucleotide which is complementary to a segment of a strand of one of the amplified nucleic acids.
The following table furnishes information about the normally used enzymes and about chromogenic substrates to be possibly used in combination with said enzymes.
After gel electrophoresis of the reaction mixture the detection of the amplification products may be performed in the gel, but also in solution or after binding to a solid matrix. A number of systems are presently available on the market, which serve the detection of amplified DNA and can be tailored to the requirements regarding the detection of T1-mRNA.
While the above-mentioned methods involve the reverse transcription and/or amplification of the nucleic acid to be detected (in the present case T1-mRNA), thereby permitting a detection of even very slight amounts of T1-mRNA, other methods are based on the detection of the molecules by amplification of the signal. An example thereof is the bDNA method (Pachl et al., 1994) in which the nucleic acid to be detected is coupled via hybridization with an oligonucleotide to a solid matrix, via a further hybridization with a second oligonucleotide to a branched DNA, which in turn hybridizes with a multitude of oligonucleotides coupled with a signal-producing enzyme. The attachment of a great amount of signal-producing enzyme units per existing target molecule is possible thanks to the branching of the DNA.
In a further embodiment of the method according to the invention the sample material is contacted with a T1-specific antibody or fragments of such an antibody. Suitable antibody fragments are e.g. Fab- and F(ab)2 fragments. The antibodies or antibody fragments can e.g. directly be incubated with the tissue section or, however, be exposed to an immunoassay in which protein extract is e.g. fixed to microtiter plates, separated on a gel matrix or made accessible to the antibody in another way or brought into contact therewith. The antibodies may be monoclonal or polycolonal antibodies; they may e.g. be mouse, rabbit or rat antibodies. The antibodies should specifically react with the T1 protein or selected epitopes of said protein. In one embodiment the method is carried out with antibodies which are specific for the p9 peptide or the p16 peptide of mouse T1 (Werenskiold, 1992). The p9 peptide derives from a region which covers a complete immunoglobulin-similar semidomain of the protein. The p16 peptide corresponds to the carboxy-terminal part of the protein and contains no sequence related to the lgC2 motif of the immunoglobulin superfamily. The antibodies can be directed against the p9 or p16 peptide of the mouse or corresponding peptides of other mammals provided that they cross-react with the corresponding T1 protein of human origin.
In a preferred embodiment the antibodies are directed against antigenic determinants of the human soluble T1 protein. To minimize the risk of a cross reaction with complete membrane-bound receptor, a peptide should be selected which is not present in the T1 receptor. In a preferred embodiment the antibody is directed against a peptide which comprises the sequence p-SKEC (SEQ ID NO:5). The region coding for said peptide is located between the exons 8 and 9 and is only expressed for the soluble protein (T1-S), but not for the receptor (T1-M).
According to the invention there is further provided a kit which is suitable for performing the method according to the invention. The kit contains T1-specific antibody or fragments thereof and optionally means for detecting the antibody or the fragments thereof. These means may for example be enzyme-conjugated anti-lg antibodies which specifically bind to the anti-T1 antibodies respectively used. Said antibodies may be detected with the standard methods that have already been discussed above. The means required therefor, e.g. enzyme substrate, may also be provided in the kit. Furthermore, the kit may be constructed such that the antibodies or antibody fragments which are suitable for detection are present in coupled form with the solid phase. The solid phase may e.g. have the form of microparticles, such as glass, polyacrylamide or Sephadex beads, or consist of microtiter plates. Other possibilities of fixing the antibodies to a solid matrix are also included.
The antibody (or fragments thereof) which is provided in the kit according to the invention may be a monoclonal or polyclonal antibody. Said antibody is produced in a manner known to the person skilled in the art by immunization with the respectively desired antigen, i.e. the p9 or p16 peptide. Sufficient amounts of said peptides can be provided by recombinant expression in eukaryotic systems, e.g. in the vaccinia virus system (Werenskiold, 1992) or in prokaryotic hosts, e.g. in E. coli, B. subtilis or streptomycetes. Instead of natural or recombinantly expressed proteins, synthetically produced peptides are used for immunization in a further embodiment. Particularly preferred are peptides which comprise the sequence p-SEKC (SEQ ID NO:5).
As has already been mentioned, the nucleotide sequence of the T1 gene and thus the coding region of the T1-mRNA are known. Being aware of the sequence, one skilled in the art can readily select suitable primers. Preferred embodiments provide for the use of an oligonucleotide with the sequence 5xe2x80x2-CTT TGA TCA CCT GAA CTT TCT CTA GCA-3xe2x80x2 (SEQ ID NO:1) as the first oligonucleotide and that of an oligonucleotide with the sequence 5xe2x80x2-AGT TTT CGG TTG TTG GTG CAT TTC-3xe2x80x2 (SEQ ID NO:2) as the further oligonucleotide. In preferred embodiments at least one of the oligonucleotides is conjugated at its 5xe2x80x2 end with an antigen or an enzyme capable of producing a signal.
In a further embodiment a kit is provided which is suitable for detecting T1-specific nucleic acids, preferably T1-specific mRNA. The kit contains at least one oligonucleotide which is complementary to T1-mRNA and can thus hybridize therewith and optionally serve as a primer for reverse transcription and/or polymerase chain reaction. Kits which are to serve the performance of a PCR in the end may further contain one or a plurality of further oligonucleotides which correspond to the sense strand of the T1-mRNA and allow for the amplification of the T1-mRNA in combination with the first oligonucleotide.
Moreover, an inventive kit for detecting the T1-mRNA may further contain the enzymes required for the reverse transcription and/or amplification, e.g. reverse transcriptase, DNA polymerase, RNase H, T7 polymerase and/or means for detecting the amplified products. The amplification products can e.g. be detected by incorporated modified nucleotides, for example digoxigenated or biotinylated nucleotides, or however by hybridization of an oligonucleotide which is or can be labeled and is complementary to the T1-mRNA or the complementary strand thereof. A further possibility of detection lies in the use of modified primers which are e.g. connected at their 5xe2x80x2 end to an antigen which is recognized by an enzyme-bound antibody. The literature gives innumerable examples of such methods of detection, which are known to one skilled in the art and determine the design of the kit in detail.
As has already been mentioned, the nucleotide sequence of the T1 gene and thus the coding region of the T1-mRNA are known. Being aware of the sequence, one skilled in the art can readily select suitable primers. Preferred embodiments provide for the use of an oligonucleotide with the sequence 5xe2x80x2-CTT TGA TCA CCT GAA CTT TCT CTA GCA-3xe2x80x2 as the first oligonucleotide and that of an oligonucleotide with the sequence 5xe2x80x2-AGT TTT CGG TTG TTG GTG CAT TTC-3xe2x80x2 as the further oligonucleotide. In preferred embodiments at least one of the oligonucleotides is conjugated at its 5xe2x80x2 end with an antigen or an enzyme capable of producing a signal.
Furthermore, the present invention relates to the use of T1-specific antibodies or fragments of such antibodies for detecting the presence of T1 protein in tissue samples, blood or serum samples of a patient with carcinoma of the breast. The T1 protein can be detected with the method of the invention. The invention also relates to the use of T1-specific oligonucleotides for detecting T1-mRNA in sample material of patients with carcinoma of the breast. The oligonucleotide used may be a sense or antisense oligonucleotide.
The figures and the following examples will explain the invention.